Epilepsy is a serious common neurological disorder, afflicting an estimated 1% of the population worldwide. Limbic epilepsy (synonyms include complex partial epilepsy, temporal lobe epilepsy, psychomotor epilepsy) is arguably the most devastating form of epilepsy in adults for three main reasons: (1) complex partial seizures constitute the single most common seizure type, accounting for approximately 40% of all cases in adults; (2) complex partial seizures are often quite resistant to available anticonvulsant drugs; and (3) an estimated 30% experience recurrent complex partial seizures despite optimal contemporary treatment (Arroyo, S. et al., (2002) Epilepsia 43(4): 437-444). These attacks induce impairment of consciousness, thereby severely limiting performance of many normal functions (e.g., driving, maintaining employment, etc.). Therapy is symptomatic. There is no effective prevention or cure, apart from surgical intervention for a minority.
Understanding the mechanisms of limbic epileptogenesis in cellular and molecular terms may lead to novel and specific therapies aimed at preventing onset and/or progression of this disorder. Extensive experimental evidence supports the assertion that the neurotrophin, brain-derived neurotrophic factor (BDNF), promotes limbic epileptogenesis by activation of its cognate receptor, TrkB. Expression of BDNF is dramatically increased following a seizure in multiple animal models (Ernfors P. et al. (1991) Neuron 7(1):165-176; Isackson P. J. et al. (1991) Neuron 6(6):937-948; Springer, J. E. et al. (1994) Brain Res. Mol. Brain Res. 23 (1-2):135-143); BDNF mRNA and protein content are also increased in the hippocampus of humans with temporal lobe epilepsy (Murray K. D. et al., (2000) J Comp Neurol 418(4):411-422; Takahashi M. et al., Brain Res 818(2):579-582). Enhanced activation of TrkB has been identified in multiple models of limbic epileptogenesis (Binder D. K. et al. (1999) J. Neurosci. 19(11), 4616-4626; Danzer S. C. et al. (2004) Neuroscience 126(4):859-869; He X. P. et al., (2002) J Neurosci 22(17): 7502-7508). Administration of BDNF and transgenic overexpression of BDNF enhance limbic epileptogenesis (Croll S. D. et al., (1999) Neuroscience 93(4):1491-1506; Xu B. et al., (2004) Neuroscience 126(3):521-531). Striking impairments of epileptogenesis in the kindling model were identified in mice carrying only a single BDNF allele, while epileptogenesis was eliminated altogether in mice with a conditional deletion of TrkB in the CNS (Kokaia M. et al., (1995) Exp Neurol 133(2): 215-224; He X. P. et al., (2004) Neuron 43(1): 31-42).
Insight into the signaling pathways by which TrkB activation promotes limbic epileptogenesis in vivo will aid in the elucidation of the underlying cellular mechanisms as well as aid in the identification of novel targets for therapy. BDNF binding to TrkB results in receptor dimerization, enhanced activity of the TrkB tyrosine kinase which results in phosphorylation of Y515 and Y816 in the intracellular domain of TrkB, thereby creating docking sites for adaptor proteins Shc and PLCγ1 respectively. Both Shc and PLCγ1 are phosphorylated by TrkB, thereby initiating Shc/Ras/MAP kinase and PLCγ1 signaling respectively. Because epileptogenesis was similar in controls and trkBSHC/SHC mutant mice, we hypothesized that PLCγ1 signaling was activated during epileptogenesis in a TrkB-dependent manner and that this activation promotes limbic epileptogenesis. Substitution of phenylalanine for tyrosine at residue 816 of TrkB (pY816 TrkB) in the trkBPLC/PLC mice selectively eliminates binding and phosphorylation of PLCγ1 by TrkB, thereby permitting study of functional consequences of TrkB-mediated activation of PLCγ1 in vivo (Minichiello L. et al., (2002) Neuron 36(1), 121-137).
A chemical-genetic approach established proof of concept that transiently inhibiting the receptor tyrosine kinase, TrkB, following SE prevented epilepsy and anxiety-like behavior in a mouse model, providing rationale for developing a therapeutic targeting TrkB signaling. Temporal lobe epilepsy is a common and devastating disorder that is often associated with pathologic anxiety. An episode of prolonged seizures (status epilepticus [SE]) is thought to promote development of human temporal lobe epilepsy years later. In search of a therapeutic, we sought to identify both the TrkB-activated signaling pathway mediating these pathologies and a compound that uncouples TrkB from the responsible signaling effector. To accomplish these goals, we used genetically modified mice and a model of seizures and epilepsy induced by a chemoconvulsant. Genetic inhibition of TrkB-mediated PLCγ1 signaling suppressed seizures induced by a chemoconvulsant, leading to design of a peptide (pY816) that inhibited the interaction of TrkB with PLCγ1. We demonstrate that pY816 selectively inhibits TrkB-mediated activation of PLCγ1 in vitro and in vivo. Treatment with pY816 prior to administration of a chemoconvulsant suppressed seizures in a dose- and time-dependent manner Treatment with pY816 initiated after chemoconvulsant-evoked seizures and continued for just three days suppressed seizure-induction of both epilepsy and anxiety-like behavior assessed months later. This study elucidates the signaling pathway by which TrkB activation produces diverse neuronal activity-driven pathologies and demonstrates therapeutic benefits of an inhibitor of this pathway in an animal model in vivo. A strategy of uncoupling a receptor tyrosine kinase from a signaling effector should prove useful in diverse diseases in which excessive receptor tyrosine kinase signaling contributes.
Receptor tyrosine kinases (RTKs) are a family of cell surface receptors, many of which are important regulators of key cellular processes including proliferation and differentiation, cell migration, and cell cycle control (Blume-Jensen and Hunter, (2001) Nature 411:355-365; Lemmon and Schlessinger, (2010) Cell 141:1117-1134. The diversity evident in the 58 distinct RTKs in humans notwithstanding, these receptors exhibit similar molecular structures with ligand-binding ectodomains, a single transmembrane domain, and a cytoplasmic component that includes the protein tyrosine kinase domain. These receptors convey signals by coupling ligand binding to the ectodomain to intracellular signaling cascades by recruiting adaptor proteins and enzymes to motifs in the cytoplasmic domain containing distinct tyrosine residues that undergo phosphorylation upon receptor activation. Mutations of RTKs resulting in excessive activation of intracellular signaling pathways cause a diversity of diseases including cancer, diabetes, and arteriosclerosis (Lemmon and Schlessinger, (2010) Cell 141: 1117-1134). This led to development of a diversity of FDA-approved drugs, most of which are small molecules that target the ATP-binding site of the kinase domain. While several tyrosine kinase inhibitors have successfully treated some human cancers, emergence of side effects due to lack of target selectivity together with emergence of drug resistance has limited success with this strategy.
The epilepsies constitute a group of common, serious neurological disorders for which no preventive or disease modifying therapy is available. Among the epilepsies, temporal lobe epilepsy (TLE) is the most prevalent and is often devastating both because of its associated behavioral disorders and its resistance to anticonvulsants (Engel J. et al., (1998), Engel J. and T. A. Pedley, eds. (Philadelphia: Lippincott-Raven), pp. 2417-2426). Many patients with severe TLE experienced an episode of continuous seizure activity (SE) years prior to the onset of TLE (French J. A. et al., (1993) Ann NeurolI 34:744-780. Because induction of SE alone is sufficient to induce TLE in diverse mammalian species (Pitkanen A., (2010) Epilepsia 51 (Suppl 3):2-17), the occurrence of de novo SE is thought to contribute to development of TLE in humans. Recent work identified a molecular mechanism required for induction of TLE by an episode SE, namely, the excessive activation of a tyrosine kinase receptor, TrkB (Liu G. et al., (2013) Neuron 79:31-38). A chemical-genetic method (Chen X. et al., (2005) Neuron 46:13-21. was used to demonstrate that inhibition of TrkB signaling, initiated following SE and continued for two weeks, prevented both SE-induced TLE and anxiety-like behavior when tested one to two months after SE (Liu G. et al., (2013) Neuron 79:31-38).
These findings provide a powerful rationale for developing a drug to selectively inhibit TrkB signaling. Difficulties in developing small molecules that selectively inhibit a single RTK led us to adopt an alternative strategy, namely to identify the downstream signaling pathway by which TrkB kinase activation produced these disorders and to develop a therapeutic targeting this pathway. This study elucidates the signaling pathway by which TrkB activation produces diverse neuronal activity-driven pathologies and demonstrates therapeutic benefits of an inhibitor of this pathway in an animal model in vivo.